This invention relates to an improved process for preparing certain water-soluble azole compounds useful in the treatment of serious systemic fungal infections. More particularly, the present invention relates to an improved process for preparing the water-soluble prodrugs having the general formula 
Wherein A is the non-hydroxy portion of a triazole antifungal compound of the type containing a secondary or tertiary hydroxy group, R and R1 are each independently hydrogen or (C1-C6)alkyl, and pharmaceutically acceptable salts thereof.
Triazole antifungal compounds are well known in the prior art. Of the several classes known, one particularly potent class contains a tertiary hydroxyl group. For example, U.S. Pat. No. 5,648,372 discloses that (2R,3R)-3-[4-(4-cyanophenyl)thiazol-2-yl]-2-(2,4-difluorophenyl)-1-(1 H-1,2,4-triazol-1-yl)-butan-2-ol has anti-fungal activity. 
The utility of this class of compounds is limited by their low water solubility. For example, the solubility of the above triazole compound in water at pH 6.8 is 0.0006 mg/mL. This greatly impedes developing suitable parenteral dosage forms.
One method of addressing this problem was disclosed in European Patent Application 829478, where the water solubility of an azole antifungal agent was increased by attaching a linked amino-acid to the azole portion of the molecule 
Alternatively, WO 97/28169 discloses that a phosphate moiety can be attached directly to the tertiary hydroxyl portion of the anti-fungal compound, e.g. the compound having the formula 
U.S. Pat. No. 5,707,977 and WO 95/19983 disclose water soluble prodrugs having the general formula 
wherein X is OP(O)(OH)2 or an easily hydrolyzable ester OC(O)RNR1R2.
WO 95/17407 discloses water-soluble azole prodrugs of the general formula 
wherein X is P(O)(OH)2, C(O)xe2x80x94(CHR1)nxe2x80x94OP(O)(OH)2 or C(O)xe2x80x94(CHR1)nxe2x80x94(OCHR1CHR1)mOR2.
WO 96/38443 discloses water-soluble azole prodrugs of the general formula 
U.S. Pat. No. 5,883,097 discloses water-soluble amino acid azole prodrugs such as the glycine ester 
The introduction of the phosphonooxymethyl moiety into hydroxyl containing drugs has been disclosed as a method to prepare water-soluble prodrugs of hydroxyl containing drugs.
European Patent No. Application 604910 discloses phosphonooxymethyl taxane derivatives of the general formula 
wherein at least one of R1xe2x80x2, R2xe2x80x3, R3xe2x80x2, R6xe2x80x2 or R7xe2x80x2 is OCH2OP(O)(OH)2.
European Patent No. Application 639577 discloses phosphonooxymethyl taxane derivatives of the formula T-[OCH2(OCH2)mOP(O)(OH)2]n wherein T is a taxane moiety bearing on the C13 carbon atom a substituted 3-amino-2-hydroxypropanoyloxy group; n is 1, 2 or 3; m is 0 or an integer from 1 to 6 inclusive, and pharmaceutically acceptable salts thereof.
WO 99/38873 discloses O-phosphonooxymethyl ether prodrugs of a diaryl 1,3,4-oxadiazolone potassium channel opener.
Golik, J. et al, Bioorganic and Medicinal Chemistry Letters, 1996, 6:1837-1842 discloses novel water soluble prodrugs of paclitaxel such as 
In pending U.S. Provisional Patent No. Application Ser. No. 60/177,169 filed Jan. 20, 2000 by our colleagues, Yasutsugu Ueda, John D. Matiskella, Jerzy Golik and Thomas W. Hudyma, the entire disclosure of which is hereby incorporated by reference, there is described the series of water-soluble prodrugs having general formula I shown below 
wherein A is the non-hydroxy portion of a triazole antifungal compound of the type containing a secondary or tertiary hydroxy group, R and R1 are each independently hydrogen or (C1-C6)alkyl, and pharmaceutically acceptable salts thereof.
The compounds of general formula I are prepared in the above application by the following reaction scheme. 
In this method, A represents the non-hydroxy portion of a triazole antifungal compound of the type containing a tertiary or secondary hydroxyl group, Pr represents conventional hydroxy-protecting groups such as t-butyl, benzyl or allyl, and R and R1 are each independently hydrogen or (C1-C6)alkyl. In a preferred embodiment, R and R1 are both hydrogen.
The antifungal compound of interest, II, is converted into phosphate ester intermediate IV by O-alkylation with chloride intermediate III in the presence of a suitable base such as sodium hydride. Ester intermediate IV is then subjected to a conventional de-protection step to remove the hydroxyl-protecting groups Pr and give end product I which, if desired, may be converted to a desired pharmaceutically acceptable salt.
The present invention greatly improves on the above process by allowing the O-alkylation step to be carried out in substantially increased yield.
The present invention represents an improved process for preparing the water-soluble antifungal prodrugs of general formula I 
above. More particularly, the present invention is directed to a process for the preparation of a water-soluble prodrug of the formula 
wherein A is the non-hydroxy portion of a triazole antifungal compound of the type containing a secondary or tertiary hydroxy group, and R and R1 are each independently hydrogen or (C1-C6)alkyl, or a pharmaceutically acceptable salt thereof, which comprises
(a) reacting a compound of the formula Axe2x80x94OH wherein A is the non-hydroxy portion of a triazole antifungal compound of the type containing a secondary or tertiary hydroxy group with a compound of the formula 
xe2x80x83in which R and R1 are as defined above and Pr represents a hydroxyl-protecting group with a source of iodide ion in an inert organic solvent and in the presence of base at a temperature of from about 25xc2x0 C. to 50xc2x0 C. to form an intermediate of the formula 
xe2x80x83wherein Pr, A, R and R1 are as defined above, and
(b) removing the protecting groups Pr from intermediate IV by conventional means to produce a compound of the formula 
xe2x80x83and, if desired, converting said compound I by conventional means to a pharmaceutically acceptable salt thereof.
The compounds of general formula I function as xe2x80x9cprodrugsxe2x80x9d when administered in vivo, i.e. they are converted to the biologically active parent azole in the presence of alkaline phosphatase.
As used herein xe2x80x9c(C1-C6)alkylxe2x80x9d refers to a straight or branched chain saturated aliphatic group having 1 to 6 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, t-butyl, n-pentyl, n-hexyl, etc.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d as used herein is intended to include phosphate salts with such counterions as ammonium, metal salts, salts with amino acids, salts with amines and salts with other bases such as piperidine or morpholine. Both mono- and bis-salts are intended to be encompassed by the term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d. Specific embodiments include ammonium, sodium, calcium, magnesium, cesium, lithium, potassium, barium, zinc, aluminum, lysine, arginine, histidine, methylamine, ethylamine, t-butylamine, cyclohexylamine, N-methylglucamine, ethylenediamine, glycine, procaine, benzathene, diethanolamine, triethanolamine, piperidine and morpholine. For the most preferred embodiment, (2R,3R)-3-[4-(4-cyanophenyl)thiazol-2-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-2-[(dihydrogen phosphonoxy)methoxy]butane, the t-butylamine and lysine salts are especially preferred as they can be obtained as single polymorph crystalline solids of high purity with good solubility and stability.
The compounds of formula I can be solvated or non-solvated. A preferred solvate is a hydrate.
A most preferred compound prepared by the present invention is (2R,3R)-3-[4-(4-cyanophenyl)thiazol-2-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-2-[(dihydrogen phosphonoxy)methoxy]butane or a pharmaceutically acceptable salt thereof. This prodrug exhibits much improved aqueous solubility ( greater than 10 mg/mL at pH 7, 5-6 mg/mL at pH 4.3) compared with the parent compound which enables it to be used for parenteral administration as well as oral administration. This compound is also stable in solution, can be isolated in crystalline form and is readily converted to parent drug in vivo.
In U.S. patent application Ser. No. 60/177,169 filed Jan. 20, 2000, the compounds of formula I are made by the following general reaction scheme. In this method, A represents the non-hydroxy portion of a triazole antifungal compound of the type containing a tertiary or secondary hydroxyl group, Pr represents a conventional hydroxy-protecting group such as t-butyl, benzyl or allyl, and R and R1 are each independently hydrogen or (C1-C6)alkyl. Most preferably, R and R1 are both hydrogen. 
To elaborate on the method, the antifungal parent compound of interest, II, is converted into the phosphate intermediate IV by O-alkylation with chloride intermediate IlIl in the presence of a suitable base such as sodium hydride, potassium hydride, sodium amide, sodium t-butoxide, potassium t-butoxide, sodium bis(trimethylsilyl)amide, potassium bis(trimethylsilyl)amide, or combinations thereof such as sodium hydride plus sodium bis(trimethylsilyl)amide. This reaction step may be carried out in an inert organic solvent such as tetrahydrofuran, methyl-tetrahydrofuran, methyl t-butyl ether, diethylether or dimethylacetamide at a temperature of from about 0xc2x0 to 50xc2x0 C., more preferably between about 20xc2x0 to 40xc2x0 C., and most preferably at about 40xc2x0 C. The most preferred base is sodium hydride and the most preferred solvent is tetrahydrofuran. The most preferred R and R1 groups are hydrogen.
Ester intermediate IV is then subjected to a conventional de-protection step to remove the hydroxyl-protecting groups Pr. The reagents used in such step will depend on the particular hydroxyl-protecting group used, but will be well known to those skilled in the art. The most preferred hydroxy protecting group is the t-butyl group which can be removed with trifluoroacetic acid, hydrochloric acid or formic acid in an appropriate inert organic solvent. The inert solvent may be, for example, methylene chloride, dichloroethane, methylbenzene or trifluoromethyl benzene. In the case of the preferred deprotection step with the di-tertiary butyl ester, it is preferred to do the deprotection step in trifluoroacetic acid in methylene chloride at a temperature of from about 0xc2x0 to 40xc2x0 C., most preferably at a temperature of about 0-5xc2x0 C.
The final product I may then be recovered and purified by conventional procedures such as reverse phase C-18 column chromatography or solvent extraction.
End product I may, of course, be converted by conventional means to a desired pharmaceutically acceptable salt as described above.
It was later discovered by the present inventors that use of purified reagent IlIl gave fairly low yields of intermediate IV (approximately 10-35% yield) in the above reaction, resulting in low overall yields of product I. However, when a source of iodide ion is added to the O-alkylation step of the above reaction, the yield of intermediate IV is unexpectedly increased up to about 90%, thus also significantly increasing the yield of final product I. It is believed that the addition of the iodide ion results in in situ formation of the corresponding iodide intermediate IIIxe2x80x2 of the formula 
and that use of this reagent results in a large increase in yield of the phosphate intermediate IV. The attempt to substitute preformed intermediate IIIxe2x80x2 directly for intermediate III in the first step of the above reaction, however, was unsuccessful due to the greatly decreased stability of iodide reagent IIIxe2x80x2 compared to the chloride intermediate III. An alternative method which was successful involves using iodine in the O-alkylation step along with chloride intermediate III in the presence of base such as NaH (which also may act as a reducing agent for the iodine). It is believed that the iodine is reduced to iodide ion which then converts chloride intermediate III in situ to iodide intermediate IIIxe2x80x2 to facilitate this step of the process. The illustrative example below shows the O-alkylation step using elemental iodine which is the preferred method of carrying out this reaction to get intermediate IV.
By forming the iodide reagent IIIxe2x80x2 in situ by addition of a source of iodide ion or by reaction of iodine and reagent III in the presence of strong base, the greatly increased yield of phosphate ester IV allows the final product I to be also obtained in greatly increased yield.
The source of iodide ion is preferably sodium iodide, but may also include lithium iodide, cesium iodide, cadmium iodide, cobalt iodide, copper iodide, rubidium iodide, barium iodide, zinc iodide and calcium iodide. About 2-3 equivalents of the iodide salt is generally used per equivalent of parent compound of formula II (Axe2x80x94OH).
When elemental iodine is used in the coupling step, about 0.1 to 1.0 equivalent of iodine, preferably 0.5 equivalent, is employed per equivalent of parent compound Axe2x80x94OH.
The bases and solvents which are used when iodine or iodide ion is used are the same as those described above when reagent III is used per se.
It will be understood that where the substituent groups used in the above reactions contain certain reaction sensitive functional groups such as amino or carboxylate groups which might result in undesirable side-reactions, such groups may be protected by conventional protecting groups known to those skilled in the art. Suitable protecting groups and methods for their removal are illustrated, for example, in Protective Groups in Organic Synthesis, Theodora W. Greene (John Wiley and Sons, 1991). It is intended that such xe2x80x9cprotectedxe2x80x9d intermediates and end-products are included within the scope of the present disclosure and claims.
It will be appreciated that certain products within the scope of formula I may have substituent groups which can result in formation of optical isomers. It is intended that the present invention include within its scope all such optical isomers as well as epimeric mixtures thereof, i.e. R- or S- or racemic forms.
The pharmaceutically active compounds of this invention may be used alone or formulated as pharmaceutical compositions comprising, in addition to the active triazole ingredient, a pharmaceutically acceptable carrier, adjuvant or diluent. The compounds may be administered by a variety of means, for example, orally, topically or parenterally (intravenous or intramuscular injection). The pharmaceutical compositions may be in solid form such as capsules, tablets, powders, etc. or in liquid form such as solutions, suspensions or emulsions. Compositions for injection may be prepared in unit dose form in ampules or in multidose containers and may contain additives such as suspending, stabilizing and dispersing agents. The compositions may be in ready-to-use form or in powder form for reconstitution at the time of delivery with a suitable vehicle such as sterile water.
Alternatively, the compounds of the present invention can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, or cream. Additionally, they may be incorporated (at a concentration up to 10%) into an ointment consisting of a white wax or soft, white paraffin base together with the required stabilizers and/or preservatives.
The compounds of the invention are useful because they possess pharmacological activities in animals, including particularly mammals and most particularly, humans. Specifically, the compounds of the present invention are useful for the treatment or prevention of topical fungal infections, including those caused by species of Candida, Trichophyton, Microsporum, or Epidermophyton. Additionally, they are useful for the treatment of mucosal infections caused by Candida albicans. They can also be used in the treatment of systemic fungal infections caused, for example, by species of Candida albicans, Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus, Coccidioides, Paracoccidiodes, Histoplasma, or Blastomyces.
Thus, according to another aspect of the invention, there is provided a method of treating a fungal infection which comprises administering a therapeutically effective amount of the compound to a host, particularly a mammalian host and most particularly a human patient. The use of the compounds of the present invention as pharmaceuticals and the use of the compounds of the invention in the manufacture of a medicament for the treatment of fungal infections are also provided.
The dosage to be administered depends, to a large extent, on the particular compound being used, the particular composition formulated, the route of administration, the nature and condition of the host and the particular situs and organism being treated. Selection of the particular preferred dosage and route of application, then, is left to the discretion of the physician or veterinarian. In general, however, the compounds may be administered parenterally or orally to mammalian hosts in an amount of from about 5 mg/day to about 1.0 g/day. These doses are exemplary of the average case, and there can be individual instances where higher or lower dosages are merited, and such dosages are within the scope of this invention. Furthermore, administration of the compounds of the present inventions can be conducted in either single or divided doses.
The in vitro evaluation of the antifungal activities of the compounds prepared by the process of the present invention can be performed by determining the minimum inhibitory concentration (MIC). The MIC is the concentration of test compound which inhibits the growth of the test microorganism. In practice, a series of agar plates, each having the test compound incorporated at a specific concentration, is inoculated with a fungal strain and each plate is then incubated for 48 h at 37xc2x0 C. The plates are examined for the presence or absence of fungal growth, and the relevant concentration is noted. Microorganisms which can be used in the test include Candida albicans, Asperigillus fumigatus, Trichophyton spp., Microsporum spp., Epidermophyton floccosum, Coccidioides immitis, and Torulopsos galbrata. It should be recognized that, as prodrugs, some compounds of the invention may not be active in the in vitro test.
The in vivo evaluation of compounds prepared by the present invention can be carried out at a series of dose levels by intraperitoneal or intravenous injection or by oral administration to mice which have been inoculated with a strain of fungus (e.g. Candida albicans). Activity is determined by comparing the survival of the treated group of mice at different dosage levels after the death of an untreated group of mice. The dose level at which the test compound provides 50% protection against the lethal effect of the infection is noted.
The compounds prepared by the present invention substantially increase the solubility of the parent triazole antifungal compound and also release the bioactive parent compound (i.e. function as a prodrug) as demonstrated in human liver S9 experiments.
In the process of the present invention the preferred formula I compounds are those wherein both R and R1 are hydrogen.
In certain embodiments of the present invention, the group A in formula I represents the non-hydroxy portion of a triazole antifungal compound of the type containing a tertiary hydroxy group. In a preferred embodiment, A can be 
wherein
R3 represents phenyl substituted by one or more (preferably 1-3) halogen atoms;
R4 represents H or CH3;
R5 represents H, or taken together with R4 may represent xe2x95x90CH2;
R6 represents a 5- or 6 membered nitrogen containing ring which may be optionally substituted by one or more groups selected from halogen, xe2x95x90O, phenyl substituted by one or more groups selected from CN, (C6H4)xe2x80x94OCH2CF2CHF2 and CHxe2x95x90CHxe2x80x94(C6H4)xe2x80x94OCH2CF2CHF2, or phenyl substituted by one or more groups selected from halogen and methylpyrazolyl.
Nitrogen containing heterocycles which R6 may represent include triazolyl, pyrimidinyl, and thiazolyl.
The term xe2x80x9chalogenxe2x80x9d as used herein includes chloro, bromo, fluoro and iodo, and is preferably chloro or fluoro, and most preferably fluoro.
Specific examples of A include, but are not limited to, the following: 
In addition to the application of the present invention to structures containing a tertiary alcohol, it should also be understood that this discovery can be applied to anti-fungal agents which contain secondary alcohols. Some examples of the non-hydroxy portion of triazole antifungal compounds of the type containing a secondary hydroxy group include, but are not limited to, the following: 